Development of new and safer whooping cough vaccines is a major priority of health policy organizations and vaccine manufacturers. Evidence accumulating from clinical trials of cellular pertussis products strongly indicates that pertussis toxin will be a necessary, and perhaps sufficient, component of any new vaccine. Anticipating the need for purer and non-reactogenic reagents, we have cloned and recombinantly expressed the genes for each of the toxin component proteins in Escherichia coli. These nonfusion recombinant polypeptides synthesized to very high levels as cellular inclusion bodies, greatly facilitating their isolation. This project involves purification and characterization of these protein antigens produced by the host system. In many instances, these expressed proteins represent up to 40% of the total cell protein; however, these proteins are often insoluble and form inclusion bodies in the E. coli cell. Various techniques were developed to solubilize these proteins. We have succeeded in purifying various versions of subunits S1, S2, and S4. Manipulation of the gene segment encoding the A protomer (S1 subunit) has permitted identification of regions possessing both immunodominant and immunoprotective epitopes as well as sites critical for enzymatic activities. We expect that our recombinant reagents will allow us to develop a stable, safe, and efficacious acellular vaccine against pertussis.